DESCRIPTION (from the application): Post-transcriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. Although PTGS occurs in diverse organisms, the cellular components of the pathway and its regulation are not well understood. The helper component proteinase (HG-Pro) of plant potyviruses has been shown to suppress PTGS in plants. Using HG-Pro as bait in a yeast two-hybrid system, we have identified a novel calmodulin-related protein (termed rgsCaM) that interacts with this viral suppressor of PTGS. rgs-CaM, like HG-Pro itself, suppresses both initiation and maintenance of PTGS. Our hypothesis is that HC-Pro-suppression of PTGS is mediated by its interaction with rgs-CaM. Here we propose to exploit HC-Pro and rgs-CaM as tools to dissect the mechanism of silencing in plants. The first four aims of the proposal focus on the role of rgs-CaM in PTGS, identifying domains of the protein required to suppress PTGS as well as those involved in the rgs-CaM/HC-Pro interaction. Several approaches will be used to interfere with expression of rgs-CaM and the effect of these manipulations on PTGS and its suppression by HC-Pro will be assayed. The third aim addresses mechanistic questions, determining how and where HC-Pro and rgs-CaM act with regard to known steps in PTGS. The last two aims focus on the role of other plant proteins in the HC-Pro-mediated suppression of PTGS. We will extend investigations of three other HC-Pro-interacting proteins using the approaches that successfully identified rgs-CaM as a regulator of PTGS. Gene array technology will be used to identify genes that are regulated in response to PTGS or its reversal by HC-Pro. Finally, we will exploit the model genetic organism Arabidopsis thalianato screen for mutants that interfere with suppression of PTGS in response to HC-Pro. The plant silencing system serves as a model to understand similar pathways in animals, and has potential to be exploited for gene therapy applications and manipulation of gene expression. Given the antiviral nature of gene silencing in plants, understanding PTGS in plants could well lead to the development of antiviral strategies in humans.